PROJECT SUMMARY Obesity-induced insulin resistance is a risk factor for diabetes and cardiovascular disease. To identify new targets for insulin-sensitizing therapies, better understanding of the currently elusive mechanisms behind obesity-induced insulin resistance is needed. Cell senescence is a stable form of cell cycle arrest characterized by several hallmarks, including mitochondrial dysfunction, DNA damage, and senescence-associated secretory phenotype. While cell senescence can exert beneficial effects in human health and disease, it plays a causative role in the pathogenesis of obesity and associated vascular complications. However, the mechanisms underlying endothelial senescence in obesity and how it impacts obesity-induced insulin resistance remain poorly understood. Long non-coding RNAs (lncRNAs) are a subgroup of non-protein coding RNA transcripts that regulate gene expression in a range of signaling pathways, and changes in their expression and function contribute to the pathogenesis of human diseases. To identify lncRNAs that regulate obesity-associated endothelial senescence, transcriptome analysis of mouse vascular endothelium revealed that maternally expressed gene 3 (Meg3) is a top differentially expressed lncRNA upon metabolic stress. Meg3 knockdown causes endothelial senescence in vitro and in obese mice, which is associated with impaired mitochondrial homeostasis and function, and an increase in mitochondrial superoxide. In obese mice, Meg3 knockdown impairs hepatic insulin signaling and induces systemic glucose and insulin intolerance. This preliminary work also identified matrin-3 as a new binding partner of Meg3. Matrin-3 is a DNA- and RNA-binding protein linked to the development of neurodegenerative disorder in humans, a disease closely associated with cardiovascular disease. Matrin-3 knockdown induces oxidative stress and endothelial senescence in vitro. The expression of either Meg3 or matrin-3 positively correlates with the expression of cyclin-dependent kinase inhibitor p16 (an in vivo marker of senescence) in human liver specimens. These observations led to the central hypothesis that endothelial cell senescence contributes to obesity-induced insulin resistance, which is controlled by Meg3 and matrin-3 through the regulation of mitochondrial function. Aim 1 will test the hypothesis that Meg3 and matrin-3 are critical regulators of mitochondrial function in endothelial cells. Aim 2 will determine the role of Meg3 and matrin-3 in endothelial senescence and paracrine function. Aim 3 will test the hypothesis that endothelial senescence contributes to obesity-induced insulin resistance. This project will uncover new roles of Meg3 and matrin-3 in regulating obesity-induced cell senescence in vascular endothelium and their effects on insulin resistance, contributing to the long-term objective to develop more effective therapies for endothelial senescence-related complications of obesity, diabetes, and cardiovascular disease.